Methods and device for glycosylation analysis

Abstract

Methods of glycosylation analysis are described in which a sample containing one or more specific oligosaccharide(s) (the analyte) is contacted with a surface on which is immobilized a glycosidase specific for the analyte and/or a specific binding partner for a product of the action of a glycosidase on the analyte. The surface may be the active surface of a biosensor, e.g., a biosensor based on the principle of frustrated total reflection.

We claim: 
     
       1. A method of assaying a sample for one or more oligosaccharides of a group of preselected analyte oligosaccharides, which method comprises (a) providing a biosensor comprising a surface comprising a plurality of discrete spatial sets of adjacent enzymatic and binding regions, wherein the enzymatic region of each set has immobilized thereon a glycosidase which hydrolyzes a preselected one of said analyte oligosaccharides to produce a known saccharide reaction product and the adjacent binding region has immobilized thereon a binding partner which specifically binds to said known saccharide reaction product and to said known saccharide reaction product conjugated to a detectable label:   (b) providing a labelled reagent comprising each of said known saccharide reaction products conjugated to said detectable label;   (c) contacting said sample with said biosensor;   (d) before, after, or simultaneously with step (c), contacting said biosensor with said labelled reagent; and   (e) measuring the amount of said detectable label bound in each said binding region as an indication of the presence or amount of each of said preselected analyte oligosaccharides in said sample.   
     
     
       2. The method as claimed in claim 1, wherein the labelled reagent is contacted with said biosensor prior to step (c). 
     
     
       3. The method as claimed in claim 1, wherein the detectable label is fluorescent. 
     
     
       4. A method of assaying a sample for one or more oligosaccharides of a group of preselected analyte oligosaccharides, which method comprises (a) providing a biosensor comprising a surface comprising a plurality of discrete spatial binding regions, each of which has immobilized thereon antibodies and/or lectins which specifically bind one of a set of known saccharide reaction products prepared by glycosidase hydrolysis of a preselected one of said analyte oligosaccharides;   (b) providing a reagent comprising a plurality of different glycosidases each of which is specific to one of said analyte oligosaccharides;   (c) contacting said sample with said reagent to form a sample/reagent mixture containing said known saccharide reaction products;   (d) contacting said biosensor with said sample/reagent mixture; and   (e) measuring the amount of said known saccharide reaction products bound in each said binding region as an indication of the present or amount of each of said preselected analyte oligosaccharides in said sample.   
     
     
       5. A biosensor having a surface upon which are immobilized, in discrete spatial regions, a plurality of different specific binding partners for products of glycosidase reactions of oligosaccharide analytes; wherein said biosensor is based on frustrated total reflection, and comprises (a) a cavity layer of transparent dielectric material of refractive index n 3 ,   (b) a dielectric base of refractive index n 1 , and   (c) interposed between the cavity layer and the base, a dielectric spacer layer of refractive index n 2 , n 2  being less than both n 1  and n 3 .     
     
     
       6. The biosensor as claimed in claim 5, in which the spacer layer has a thickness of from about 200 nm to 2000 nm, and the cavity layer has a thickness of from 10 to 200 nm. 
     
     
       7. The biosensor as claimed in claim 5, in which the cavity layer has a thickness of 30 to 150 nm and comprises a material selected from zirconium dioxide, titanium dioxide, tantalum oxide, and aluminum oxide, and the spacer layer has a thickness of 500 to 1500 nm and comprises a material selected from magnesium fluoride, lithium fluoride, and silicon dioxide, the choice of materials being such that the refractive index of the spacer layer is less than that of the cavity layer. 
     
     
       8. A biosensor having a surface upon which are immobilized, in discrete spatial regions, a plurality of different glycosidases, wherein said biosensor is based on frustrated total reflection, and comprises (a) a cavity layer of transparent dielectric material of refractive index n 3 ,   (b) a dielectric base of refractive index n 1 , and   (c) interposed between the cavity layer and the base, a dielectric spacer layer of refractive index n 2 , n 2  being less than both n 1  and n 3 .